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was an amateur microscope maker
By looking at a piece of cork, he discovered cells
Coined the term cells based on where they had been, he wasn’t seeing actual live cells |
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was a microscope designer and he was the first to see live cells
At this point we had never seen dividing so we didn’t know where they came from and much more |
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found that nucleus were important in plant cells, and that was later found for animals
He also found that there was movement in cells, cytoplasm was active, thus they contained living material |
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| Found cell theory- cells are the basic unit of life |
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Question was where do cells come from?
He claimed all cells derive from cells
Also recognized that many human diseases are problems with cells |
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| Did an experiment that showed living organisms don’t arise spontaneously |
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• Plasma membrane
• Metabolism
• Capacity of reproduction
• DNA, RNA, protein, ribosomes |
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• Prokaryotes (Bacteria)
o Kinds
• Cyanobacteria
• Archea |
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o Typically 1-10 microns long
o Have few or no organelles
o DNA is circular and in cytoplasm
• Because they were around prior to development of nuclei
o RNA and protein synthesized in same compartment
o Don’t have the movement in the cytoplasm like eukaryotes
o Divide by binary fission
o Mainly unicellular |
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• Animals
• Plants
Cell wall
Vacuoles
Chloroplasts
Most cells don’t move
• Fungi
• Protists – unicellular eukaryotes |
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| Eukaryote characteristics |
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o Cells are generally 10-100 microns long
o Lots of organelles
o DNA is linear in the nucleus
o RNA in nucleus while protein is in cytoplasm
o Have a cytoskeleton in cytoplasm creating motility in and of cells
• Cytoplasmic streaming
• Endocytosis and exocytosis
o divide by mitosis and meiosis
o both unicellular and multicellular |
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| allows selective permeability |
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| Have double lipid bilayer |
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| site of some protein synthesis |
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stacks of discs, primarily for sorting proteins to different parts of the cell
• lots of post-translational modifications here |
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| have digestive enzymes, site of breakdown |
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mainly break down fatty acids molecules
• named after hydrogen peroxide |
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| is the fluid in the cell, while the cytoplasm is the fluid and the organelles (excluding the nucleus) |
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| involved in protein synthesis |
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| cytoskeleton has three parts: |
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o Microtubules
o Microfilaments
o Intermediate filaments |
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o Made up by centriole and peri-centriolar material (shit around the centriole).
house microtubules |
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• Discovered mitochondria might be from symbiotic relationship of bacteria in eukaryotes
o Both same size (and both circular?)
o Both divide by binary fission
o Both have double membrane
o Mitochondria have DNA
• That was weak evidence, the stronger is
o Mitochondria have own ribosomes which resemble bacterial ribosomes
o There are slight differences in the genetic code for the mitochondrial genome
• there are 3.4 billion base pairs, 23,000 genese in nucleus while only 16,000 in mitochondria, 37 genes
• UGA is stop codon in nucleus but is tryptophan in mitochondria, this is often the case in many organisms
This suggests a common origin
Also some bacteria can use UGA for tryptophan
o the sequence of the genes of mitochondria resemble bacterial codes more so than nuclear like |
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have shapes to increase surface area (microvilli)
also have intercellular junctions |
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| star like in extracellular matrix |
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| good colonies for observation |
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• good things to observe
• drosophilia
• C elegans
• Mouse
• E. Coli
• Yeast
• Arabidopsis |
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| minimum distance between objects that can be distinguished |
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| Lenses expensive in order to deal with: |
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Spherical abborations: curve shape of lenses can only focus in middle not edges or vica versa at once since specimen is flat
chromatic abborations: different colors are bent different so around each objects would be a halo
• to solve these problems they use a series of lenses and thus why there is variance in price |
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| Types of light microscopy (first 3) |
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Definition
• conventional wide field-
• phase contrast- wiki it
• darkfield – wiki it
o makes it easy to count individual cells but not that detailed
• these first three require staining
o some textile dyes that are used are hematoxlyn which binds to negatively charged molecules
o some enzymes are used on substrates to make it be able to be colored and insoluble products
• ex. Alkaling phosphatase on substrate BCIP makes it very blue and insoluble and by doing this we could easily see which is intestinal tissue because there is a lot of BCIP in intestinal tissue |
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• kill the cell
• permeabalize it so molecules and stains can go in and out
• cross link so structures stay in place
• ex. Gluteraldehyde which can act with two different things at once holding together |
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| use special machine called microtome |
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o there is difference intensities for different wavelength
o absorbs at one wavelength and emits at another
• ex. Absorbs excited blue wavelength and then emitted as green |
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somewhat similar to light microscopy but is at a perpendicular angle, and a mirror that reflects blue wavelengths and allow others to go through (dichroic) and if the specimen has fluorescein it will emit green light
• it also has a black background and makes it really easy to see small amounts of fluorescein, or many other dyes like it
DAPI which binds DNA and fluoresces, this absorbs ultraviolet light and emits
• Around every source of light is a hazy glow since it bounces of other items near it |
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uses a laser to get a point of light at a time and is scanned across the specimen so we are only eliminating one dot at a time
• This pinhole is in a confocal plane and the one near the projector eliminates a lot of the fuzz making it more detailed
• This is called laser-scanning confocal microscopy
• How do you get a tag onto a certain part of the cell
• With black background we can detect tiny amounts of fluorescence
• When we see a pin of light at about 200 nm but it interference around and light is seen around the disk- this disk is airy disk
Result of this is when we have 2 disks next to each other, it is observed as one big disk
Thus when shapes are smaller than the 200nm resolution, it is impossible to see using light so we use something else |
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o They wanted to top this light problem
o They used a molecule that was photo-activatable that fluoresced kind of like fluorine but not until it was activated with another wavelength
• This time it had to be hit with UV light first before luminating unlike when it was visual light before
o By finding this, a low level UV light will only turn on some of the molecules that are very close and no longer was blurry if some where on but not all
o Photo bleaching is when they were turned off so we activate the bleach, image and do it again thus building up an image
o if we saw one disk then it is very easy to find the center and we could have a computer put just the center so if repeated a lot, we can get just the centers
o this whole process is called photo-activated localization microscopy or PALM
o this improves the visible area from 200nm to about 25nm
o its not very fast so if something is moving quickly then we cant usually capture it on PALM |
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| direct immunofluorescence |
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o we can insert something into a rabbit or something that makes it create antibody and then we could attach flurophore to it and then we can see where the protein is in the body or cell
ex. green fluorescent protein can be inserted after a protein and will usually be transmitted to new cell |
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| indirect immunofluorescence |
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Definition
since it is pretty weak light so we have antibodies attach to the main one and these result in more fluorophores and thus much brighter signals
o the main antibody is primary and others attached is secondary, however many
o often not used in living cells because they combined antibodies may hinder their function
o if we wanted to use in living cells we can use GFP which absorbs blue light and emits green light, so people have taken this gene and put into other systems so we can see fluorescent parts
o using GFP, we can put next to a gene we want and it doesn’t alter the protein production
• then using epifluoresence or confocal we shine blue light on it and see green where the protein it
o someone created a photo-activatable GFP so it wouldn’t shine until UV was shined on it |
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• If light microscope is limited to wavelength of light, we want something with a smaller wavelength, electrons
• They discovered electromagnets can focus a beam of electron, and these microscopes found in 30s |
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| Transmission electron microscopy (TEM) |
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o Done in a vacuum so it doesn’t bump into air molecules and very high voltage as well, and water must be eliminated so prevent looking at that
o Uses thin sections 50-100 nm thick
o Fixatives are used, a typical one is OsO4, and since these have more than one reactive site they are cross linkers between objects in cells, this one having 4. This binds great to membranes so they are generally darker
o All these reasons show that cells are DEAD
o Beam of electrons that go through lenses and projected onto a screen of photographic film
o The image is created by the difference in what deflects and what passes through
o Its hard to see the surface topologies and thick detail |
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| Scanning electron microscopy (SEM) |
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o The specimen is coated in heavy metal to deflect electrons thus a detector reads all the directions electrons are deflected and can make a topology of it
• There are tricks to get a TEM to show some topology since it has much better resolution that SEM:
metal shadowing
freeze fracture
freeze etch
negative staining |
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o We could coat the specimen on one side with heavy metal and thus only mark on one side of the material
o We then add carbon from above which creates a not electron dense coating
o Then can float in organic solvent so the cell is washed away and just the replica is read by TEM |
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o If we wanted to see the surface of a nucleus,
o we could start by freezing in a block of ice
o Then crack and hope it breaks along a membrane which they tend to do
o This could then be used for metal shadowing
o Finally TEM it |
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o If we wanted to see stuff not on surface but deeper
o Freeze
o Crack
o Sublimate which etchs away some
o Metal shadow
o TEM |
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o Place macromolecule on a surface
o Wash with heavy metal and then wash most off so it ends up in crevices between molecule and surface
o Then electron hits of the metal and this is used to create an image
o Essentially is imaging the spaces left around the macromolecule |
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| Isolation and Growth of cells |
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• Direct manipulation of cells
o We find one cells tells the other what to do
• We know in C. Elegans, one region of cells from embryo make up digestive track and we know the cell next to it tells it to do so. This happens between there being 4 and 8 cells
o If we know this we can take it out and grow on its own, this called primary culture
o Yet if isolated on own it wont grow the digestive track cuz it doesn’t get signaling
o Cell lines divide indefinitely while most human cells divide 50 times and then quit
o Cancer is an example of indefinate divided, HeLa cells derived from human cervical cancer in 1952 and is the first cell lines |
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| ways of breaking up cells |
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sonication- use of high frequency sounds
mild detergents- to break through membranes; force through small hole or space |
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o Separates the different components by spinning them quickly
o Three methods:
• Differential centrifugation
• Velocity sedimentation
• equilibrium sedimentation |
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| Differential centrifugation |
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Centrifuge broken cells at low speed at about 1000 times force of gravity for 20 mins and whole cells that didn’t break, a long with other large structures go down
Take this out and centrifuge again faster, pellet contains mitochondria, lysosomes, peroxisomes
Take out and spin faster, we can get out microsomes and small vesicles
Very high speed (150,000 times gravity for 3 hours) gets out large macromolecules, ribosomes, virus |
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the sucrose helps stabilize against convective mixing so different bands appear, different components go different distances
to collect, fractionate by making hole in bottom of tube and collect stuff at different rates
different sized molecules stabilize at different levels in the test tube |
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| equilibrium sedimentation |
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separates depending on buoyant density, not paying attention to size and shape
incredibly sensitive, can even separate things that have different isotopes but also makes bands of different components |
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| • 95% of dry weight is C= 50%, O= 20%, H=10%, N=10%, P= 4%, S= 1%, |
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o Methyl (-CH3) NP
o Hydroxyl (-OH) P
o Carboxyl (-COOH) P/I
o Amino (-NH2) P/I
o Phosphate (-PO4) I
o Amide (- CO-NH2) P
o Sulfhydryl (-SH) P |
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| Four main classes of small molecules |
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o Amino Acids
• Has amino group, carboxyl group, side chain R
• These strung together make up proteins
o Nucleotides
• There is a base, sugar, phosphate
• Strung together make nucleic acids
• Has the deoxy group in DNA
o Sugars
• Combine to make up polysaccharides
o Lipids
100-1000 MW |
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• Polypeptides
o Created by combination of amino acids
o Have amino and carboxy terminus
• N terminus is Amino
• C terminus is Carboxyl
o The 4 side groups of the polypeptide have different characteristics
o Lots of possible combinations from even 5 amino acids, but only some are possibly stable
1000-1000000 MW |
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• Hydrogen Bonds
o When water molecules are nearby they tend to align because of the partial positive charge of one hydrogen bonds to the negative charge of the oxygen
o 1/20 the strength of a covalent bond
• Hydrophobic interactions
o Polar molecules and ions interact with water very well dispersing equally throughout
o Nonpolar molecules do not do this but instead bond well with other water molecules and exclude the nonpolar, thus pushing the water together and the nonpolar does not dissolve, this is hydrophobic
o An example is fats
o Molecules with hydrophobic and philic parts are amphipathic
• These molecules line up so hydrophilic is in water and hydrophobic sticks out
• Or they also form small structures in the water where the hydrophobic groups are hidden from the water
• Ionic bonds
• Van der Waals Interactions
o The maximum distance where molecules attract is called the van der waals force equilibrium point
o very weak but if we have two proteins whose faces fit into one another, the forces can be pretty strong
• Brownian motion is atoms always colliding with other molecules like proteins and the smaller they are the more they bounce around. This is important on small scale cuz it has a stronger effect |
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• if we put a protein in a solution with some salts, it will fold and conform meaning that it does it on its own
• disulfide bonds are important because they form in extracellular environment (not cytosolic compartments such as in organelles in the cell), but they stabilize protein conformation, but they do not affect what shape it will be
• common folding patterns
o a-helix
• as long as side chains don’t get in the way, a molecule can get in this formation because it only needs to involve atoms of the polypeptide backbone
o b-sheet
• same as a-helix regarding formation |
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| Protein levels of organization |
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o primary
• sequence of amino acids
o secondary
• structure of small continuous sequence like a-helix or b-sheet
o tertiary
• refers to the structure of an entire polypeptide chain or of one domain (a part that can fold on its own) of a protein
o quaternary
• refers to the structure of protein complexes such as proteins interacting with others |
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| methods for determining protein structure |
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Definition
o x-ray crystallography
• get protein and purify it and if lucky it will crystallize and put beam of electrons through it and look at defraction that will tell you the structure of the protein
• William Lawrence Bragg figured out one could use electrons like this but used it for not proteins 1912
• Dorothy Hodgkin- 1930s- she was the first to successfully do it on protein structures
o NMR spectroscopy
• Get a pattern of how hydrogens interact in the structure and we can paint a picture of what the structure should look like |
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• First discovered in 1904 when a student only had half the number of blood cells and saw the freak shapes
• In the beta chain of hemoglobin, it usually is glutamic acid but a mutation results in coding for valine instead which goes from hydrophilic to hydrophobic
The sickle version results in sticky ends on the beta chains which is not a problem if it is oxygenated
Deoxygenated normal red blood cells cause indentations in the beta chain
Deoxygenated sickle cells also get these indentations, but these sticky ends they have fit into the indentations of neighboring hemoglobin and creates a chain of them resulting in the sickle cell
• Sickle cell shape can interfere with blood flow
• 1949 is when we understood this and is probably when we first understood a disease the molecular level
• ways to fight it
gene therapy
replace the cell line that is creating the blood cells
o we know the genes of about 3000 diseases
o at least double that for where we know the loci of gene area but not exactly which |
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| • Depend on binding to other proteins and molecules |
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• A molecule that binds to the binding site of a protein
• Is binding by weak noncovalent bonds
• It can be on a specific continuous stretch of a sequence or it can bind to noncontinuous parts of a sequence
An example of the latter is antibodies |
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o Catalyze the conversion of substrates into products by binding to the substrate and encouraging some sort of reaction to occur quicker than it would otherwise
o Binding site = catalytic site
o Allosteric enzymes
• Whose conformation is determined in part by binding ligands at sites other than the catalytic site
Ex. An enzyme binds ADP at another site and puts the protein into an active conformation so it can catalyze a reaction with something else
• Two really common processes
GTP binding and hydrolysis
• When GTP is hydrolyzed, one phosphate is released and is bound GDP which isn’t holding on to helix any longer
• GTP results in binding of tRNA and hydrolysis results in release of tRNA
Phosphorylation
• Kinases catalyze adding a phosphate group and phosphotase removes the phosphate group
• This is often done on serine, threonine, tyrosine because they are 3 amino acids with OH on them so it is easily done |
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o Binding ATP results in changing of conformation which is toggling between the binding and unbinding yet the next step where it is hydrolysized is almost irreversible and it goes on before it finally releases ATP
responsible for moving of molecules and generate the forces responsible for muscle contraction |
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• Fluid mosaic model, about 5 nm thick, hydrophobic region being combined about 2.5 nm
• molecules have a hydrophobic and hydrophilic end and often result in a micelle where hydrophilic ends faces out and phobic stays on inside
• in bilayer, molecules create bilayer because one end is philic other phobic so phobic stick together and philic is on outside ( See first figure on PDF)
o molecules with both are amphipathic |
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| is shown as the lipids in PDF with hydrophilic heads and hydrophobic bodies |
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1925
o was done where the took RBC and lysed them so cytoplasm spilled out
o then fractioned them so they got only the plasma membrane of the red blood cell (RBC ghost)
o took these ghosts and dispersed them on the surface of water and set up so they could move one edge of the container until the ghosts were all packed tightly, measuring the force required, and at a certain point there was a resistance to the force, this being when they reach twice the surface are of the ghosts.
o Since amphipathic they align in water a certain way based on phobia and philia |
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Definition
o The turn in the lipid is a result of a double bond
• These molecules are unstaturated
• Polyunsaturated fats means there are a lot of place of unsaturation
o If more lipids don’t have this and are just straight would result in a lot more lipids able to pack in and thus less fluid
o The turns thus result in more fluidity
o Important because:
• Fluidity of the bilayer is important for proteins interacting with each other
• Also important because lipids and proteins added to certain places but need to be distributed all over
• places where membranes are fusing together and the fluidity always membranes to mix
• Each cell division requires that the membrane components are equally distributed |
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o Very large difference between the cytosolic face and non-cytosolic face of the bilayer
• This is always certain way for cell membrane as well as vesicles or bilayers within the cell have cytosolic face
o Exocytosis would require a flip of these membranes to make sure it is correct in both extracellular as well as when in cytoplasm and this is done by proteins called flippases
• Flippases can flip either way
o Proteins or phosholipids with sugar groups CANNOT be flipped |
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• How proteins associate with the bilayer
o 1-3 are transmembrane proteins as they are crossing through in different ways or different kinds of proteins
• 1 and 2 are most common ways to see proteins pass through, that being as alpha helix
• a turn is about .54 nm and 4 amino acids, thus the depictions are pretty accurate, indicating about 20-22 amino acids are in the hydrophobic region which would also be nonpolar
o 4 is stuck within the lipid bilayer
o 5-6 are covalently attached to the lipids thus associating them to the membrane
o 7-8 shows proteins attached to other proteins associated with membrane in some way
• attached by weak bonds which can be affected/broken by
ionic strength increase
change in pH
change in temperature
• from this 7 and 8 would be broken but all the others would not be broke since they are covalently attached and not by weak bonds
o integral membrane proteins 1-6
o peripheral membrane proteins 7-8 |
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| Studying Membrane Proteins |
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Definition
• if you want to find the free energy (look at transfer free energy for amino acids) required to transfer a stretch of 20 amino acid protein to water we would see a graph (that went from n terminus to c terminus) that was low and had a big curve in middle and then went back down
o the point where it crosses 20 kCal/Mol makes us think it is a transport protein
o this plot is referred to as a hydropathy plot |
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o take RBC, lyse, collect only plasma membrane, wash and reseal the ghosts in normal orientation or inside-out
o do experiments with A) leaky ghost B) normal and C) inside-out
o do gel electrophoresis on these fuckers
• A) increase salt to break weak bonds, centrifuge to separate pellet and supernatant
We would see 2 in pellet and 5 in supernatant
Those that fell of from salt would be in supernatant (cuz the weak bonds were broken thus peripheral)
• Take A B C and label the proteins with protein dye that is impermeant that will go all around leaky ghost inside out, but will be only on outside of B and C
A) everything would be labeled
B) two bands would appear where the pellet from A were telling us part of protein must be on outside face of the cell
C) everything would be labeled showing that these are on the outside as well as the two on the inside. This shows the other 5 were peripheral on the inside
• Label A B C with membrane-impermeant carbohydrate die
A) would have two in pellet spot
B) same two in pellet spot
• Sugar groups on the outside of the membrane
C) would have nothing |
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